Applying Ultrasound to Mechanically and Noninvasively Sensitize Prostate Tumors to TRAIL-Mediated Apoptosis in Vivo

Introduction: : The King Lab has previously demonstrated how fluid shear stress sensitizes breast, colon, and prostate cancer (PCa) cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis through Piezo1 activation of the intrinsic apoptotic pathway [1]. Piezo1 is a mechanosensitive ion channel (MSC) that is activated by external stimuli, triggering calcium influx to promote pro-apoptotic pathways in cancer cells (Fig. 1A) [1,2]. We aimed to explore the biochemical response that occurs in PCa when treated with safe doses of therapeutic, focal ultrasound (FUS) combined with TRAIL. TRAIL binds to death receptors 4 and 5 (DR4/5) on cancer cells, while sparing healthy cells [3]. Soluble TRAIL alone lacks efficacy in humans to be used as a cancer therapy, and thus has been in and out of clinical trials.

Materials and

Methods: : TRAIL sensitization via FUS was examined in weakly metastatic LNCaP and highly metastatic PC3 PCa cells. The FUS system and parameters are depicted in Figure 1B and Figure 1C, respectively. PC3 cells were treated with TRAIL (200 ng/mL) before FUS treatment (Fig. 1D,E). A Piezo1 inhibitor (GsMTx-4), calcium chelator (EGTA), and PIEZO1 knockdown cell line were used to confirm MSC activation during FUS+TRAIL treatment. After 24h, apoptosis was assessed using the Annexin-V/propidium iodide (AV/PI) assay and mitochondrial depolarization was measured using a JC-1 assay via flow cytometry. TRAIL sensitization was determined from the viability measured in the AV/PI assays, comparing the untreated conditions to the TRAIL-treated conditions (Fig. 1E). An in vivo study was conducted in which subcutaneous PC3 tumors were inoculated on the flanks of eight-week-old, male NU/NU mice (Fig. 1F,G). TRAIL (2 mg/kg) was injected intratumorally, followed by a single FUS treatment (10 min/tumor), using the same pulse repetition frequency, duty cycle, transducer and burst duration as in Figure 1C at 944 kPa. Two treatment doses were administered ~one week apart, and tumor volume was measured over time via calipers. Tumors were processed by the Translational Pathology Core at Vanderbilt for immunohistochemistry (IHC) analysis upon resection.

Results, Conclusions, and Discussions:: Combination treatment showed a pressure-dependent increase in TRAIL-mediated apoptosis and mitochondrial depolarization for PC3 cells, consistent with an increase in TRAIL sensitization (Fig. 1D,E). Knockdown of PIEZO1 in PC3 cells reduced TRAIL sensitization upon combination treatment. Similarly, treatment of LNCaP and PC3 cells with GsMTx-4 and EGTA reduced TRAIL sensitization, indicating FUS-induced activation of pro-apoptotic pathways. Apoptosis array results from a proteome profiler yielded consistent results, such as enhanced cleaved-caspase 3 (CC3) and cytochrome c expression for the FUS+TRAIL treatments.

The in vivo study demonstrates, for the first time, the use of FUS to decrease tumor burden when applied with soluble TRAIL (Fig. 1F,G), evident by significantly decreased tumor volume for the combination treatment compared to PBS-, TRAIL-, or FUS-only treatments. Various intervals between the TRAIL injection and FUS exposure were also tested (0-4h), with the 0h interval showing the most efficacy towards reduing tumor burden. IHC analysis of the tumors revealed upregulated Piezo1 expression for the FUS+TRAIL-treated tumors comparatively. Enhanced cell death was observed via CC3 and TUNEL staining for the FUS+TRAIL combination therapy, further supporting successful tumor cell ablation following treatment. No significant correlation between Piezo1 expression levels and tumor volume was observed, but the trend suggests an inverse relationship.

TRAIL and FUS combination therapies provide a localized, non-invasive mechanobiology-based treatment, employing technology already widely available. In this study, most experiments were performed at or below 944 kPa, at which the mechanical index (MI) is 0.9. An MI of 1.9 is the limit considered safe by the FDA before any major cavitation is expected to occur; higher values of MI increase the probability of mechanical damage [4]. This is critical considering that the prostate is located near neurovascular bundles that risk being damaged during invasive treatments options [4]. Future work will apply these studies to additional cancer types and combine FUS with additional chemotherapy regimens.

Acknowledgements and/or References (Optional):: This research was funded by the NSF-GRFP.

References: [1] JM Hope, Cell Death & Dis, 2019, 10, 1-15; [2] JA Dombroski, Cells, 2021, 10, 1-14; [3] AR Fabiano, Adv. Science, 2025, 12, 2412995. [4] Şen, T., Tüfekçioğlu, O. & Koza, Y. Anatol J Cardiol, 2015, 15, 334–336; [4] Y Wu; Nature Biomed. Engr, 2021, 5, 1336-47.